Aspergillus fumigatus is the major causative agent of invasive aspergillosis, a severe and life-threatening disease of immunocompromised patients. A. fumigatus is a ubiquitous and adept pathogen, able to adapt quickly to the mammalian lung environment and to effectively handle the stresses encountered while growing within the human host. Because of the multi-factorial nature of fungal growth, attempts to identify individual A. fumigatus virulence determinants are often unsuccessful. However, previous studies have consistently proven that filamentous fungal pathogens must be able to coordinate proper hyphal morphogenesis in order to invade tissue and cause high mortality rates. Unfortunately, our knowledge of the molecular mechanisms supporting sustained polarized growth and the establishment and progression of invasive disease is incomplete. To elucidate these mechanisms, better models of the signaling pathways and protein interactions that regulate fungal morphogenesis must be developed. Ras proteins orchestrate multiple fungal morphogenetic processes in pathogenic fungi. Regulation of these processes by Ras plays an essential role in fungal virulence, making fungal Ras signaling an invaluable tool for probing fungal pathogenesis and identifying new targets for novel therapeutics. Although many aspects of Ras signaling pathways are often considered too highly conserved to serve as antimicrobial targets in eukaryotic pathogens, we have identified novel, fungal-specific protein domains that define fundamental differences between fungal and human Ras proteins. These include two areas of significant divergence between fungal Ras proteins and their human counterpart, H-ras: 1) the Invariant Arginine Domain (IRD), a novel domain conserved in the RasA homologs of every available fungal genome but not present in H-ras, and 2) an extended hypervariable region (HVR) in filamentous fungi. Our preliminary data show that the IRD and HVR are required for Ras function during hyphal growth and morphogenesis, revealing fungal-specific aspects of Ras signaling. The overall objectives of this proposal are to fully define the impact of these fungal-specific protein domains to Ras biology, using mutations of these domains to identify novel Ras pathway interactions critical for polarized morphogenesis and virulence. Using mutational and biochemical analyses, Aim 1 or this proposal will define the contribution of the IRD and HVR to A. fumigatus Ras biology. These studies will address how mutation of the IRD and HVR affect RasA GTPase activity, activation, localization and interaction downstream effectors. Complementing our Aim 1 studies, Aim 2 utilizes an unbiased proteomics screen to identify novel, fungal-specific Ras interactions contributing to Ras-mediated growth and virulence. Because Ras signaling is essential for fungal virulence, identification and characterization of fundamental differences between human and Ras pathways carries the long-term potential of uncovering novel antifungal therapies.